PROJECT SUMMARY The objective of this project is to understand mechanisms by which immature egg cells, or oocytes, become developmentally competent. Mammalian oocytes are stored in the ovary, arrested at meiotic prophase, for extended periods of time (decades in women). Following puberty, oocytes enter a period of growth in which they synthesize proteins needed for the maturation process. They are stimulated to mature by signals from the pituitary. Oocytes undergo many cytoplasmic changes during oocyte maturation, the period between prophase I and metaphase II. These include a dramatic reorganization of the endoplasmic reticulum (ER) in which the ER becomes concentrated in the egg cortex. Ca2+ uptake into the ER also occurs, and that is necessary for the egg to release intracellular Ca2+ from the ER at fertilization, a critical event required for early embryonic development. Oocytes also undergo changes that permit them to undergo cortical granule exocytosis at fertilization, an important event that renders the fertilized egg impermeable to more than one sperm and thus serves as a polyspermy prevention mechanism. Many questions remain about these events. Aim 1 will investigate whether changes in ER structure and the formation of ER-plasma membrane (ER-PM) contacts are required for developmental competence, as well as mechanisms that regulate ER reorganization, formation of ER-PM contacts, and Ca2+ uptake during maturation. Aim 2 will identify proteins that are needed for constitutive exocytosis as well as the Ca2+-regulated exocytosis of cortical granules. These experiments will make use of protein degradation methods to study the role of specific proteins, alone or in combination with other candidate proteins, using isolated or follicle-enclosed oocytes. Importantly, we will use a newly developed method for the rapid and acute degradation of endogenous proteins. Oocytes will be injected with fluorescent molecules that allow the visualization of the ER and ER-PM contacts, or with antibodies/siRNAs/morpholinos to specifically deplete proteins. The conclusions reached from these studies will be applicable to understanding oocyte development in women. Currently, the ability to mature human oocytes in vitro is an area of high importance, but methods for maturing oocytes are imperfect. A complete knowledge of all aspects of oocyte development could lead to improved culturing methods that will increase the success of assisted reproduction.